European Journal of Human Genetics (2016) 24, 78–85 & 2016 Macmillan Publishers Limited All rights reserved 1018-4813/16 www.nature.com/ejhg

ARTICLE Double SMCHD1 variants in FSHD2: the synergistic effect of two SMCHD1 variants on D4Z4 hypomethylation and disease penetrance in FSHD2

Marlinde L van den Boogaard1, Richard JFL Lemmers1, Pilar Camaño2, Patrick J van der Vliet1, Nicol Voermans3, Baziel GM van Engelen3, Adolfo Lopez de Munain2, Stephen J Tapscott4, Nienke van der Stoep5, Rabi Tawil6 and Silvère M van der Maarel*,1

Facioscapulohumeral muscular dystrophy (FSHD) predominantly affects the muscles in the face, trunk and upper extremities and is marked by large clinical variability in disease onset and progression. FSHD is associated with partial chromatin relaxation of the D4Z4 repeat array on chromosome 4 and the somatic expression of the D4Z4 encoded DUX4 gene. The most common form, FSHD1, is caused by a contraction of the D4Z4 repeat array on chromosome 4 to a size of 1–10 units. FSHD2, the less common form of FSHD, is most often caused by heterozygous variants in the chromatin modifier SMCHD1, which is involved in the maintenance of D4Z4 methylation. We identified three families in which the proband carries two potentially damaging SMCHD1 variants. We investigated whether these variants were located in cis or in trans and determined their functional consequences by detailed clinical information and D4Z4 methylation studies. In the first family, both variants in trans were shown to act synergistically on D4Z4 hypomethylation and disease penetrance, in the second family both SMCHD1 function- affecting variants were located in cis while in the third family one of the two variants did not affect function. This study demonstrates that having two SMCHD1 missense variants that affect function is compatible with life in males and females, which is remarkable considering its role in X inactivation in mice. The study also highlights the variability in SMCHD1 variants underlying FSHD2 and the predictive value of D4Z4 methylation analysis in determining the functional consequences of SMCHD1 variants of unknown significance. European Journal of Human Genetics (2016) 24, 78–85; doi:10.1038/ejhg.2015.55; published online 18 March 2015

INTRODUCTION facilitates the production of stable DUX4 mRNA due to the presence Facioscapulohumeral muscular dystrophy (FSHD; OMIM 158900) is a of a polymorphic DUX4 polyadenylation signal distal to the D4Z4 common myopathy in adults, with a recently reported prevalence of repeat array.8,13 D4Z4 chromatin relaxation on non-permissive ~ 1:8000.1 FSHD is clinically characterized by weakness of the facial, chromosomes lacking a DUX4 polyadenylation signal do not cause shoulder girdle, trunk and upper arm muscles, which can be FSHD in the absence of detectable levels of DUX4 mRNA.8,14 asymmetric, and progresses to involve humeral, anterior lower leg Autosomal dominant FSHD1 is the most common form of FSHD muscles and pelvic girdle muscles.2 The onset of the disease is typically (495%), in which D4Z4 chromatin relaxation and DUX4 expression in the second decade of life, but the disease progression and severity are caused by a contraction of the D4Z4 repeat array to a size of 1–10 are highly variable.3 units.15,16 In the uncommon form of FSHD (FSHD2), D4Z4 The genetic forms identified thus far, FSHD1 and FSHD2, are chromatin relaxation occurs in the absence of D4Z4 repeat array clinically indistinguishable.4 Both forms are associated with partial contraction.5 In FSHD1, chromatin relaxation and CpG hypomethyla- chromatin relaxation of the D4Z4 macrosatellite repeat array on the tion are restricted to the contracted allele, whereas in FSHD2 subtelomere of the long arm of chromosome 4 and transcriptional chromatin relaxation and CpG hypomethylation occur at the D4Z4 derepression of the D4Z4 unit-encoded DUX4 retrogene in skeletal repeat arrays of both copies of chromosome 4 and in the highly muscle.5–9 DUX4 is a germ line transcription factor that is normally homologous repeat arrays on chromosome 10.4,9 repressed in somatic cells.7 Its expression in skeletal muscle activates Heterozygous variants in the structural maintenance of chromosomes genes involved in germ line and early stem cell development, as well as flexible hinge domain containing 1 (SMCHD1) gene on chromosome 18 specific classes of repeat elements, and overexpression of DUX4 in account for the majority of FSHD2 cases.14 SMCHD1 is an atypical somatic cells causes cell death.10–12 member of the SMC gene superfamily, a family of proteins which is To cause FSHD, D4Z4 chromatin relaxation must occur on a involved in chromosome condensation and cohesion, genome main- specific genetic background of chromosome 4 (most often 4A161) that tenance and gene regulation.17–19 Chromatin immunoprecipitation

1Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands; 2Neurosciences, BioDonostia Health Research Institute, Hospital Donostia, San Sebastián, Spain; 3Department of Neurology, Radboud University Medical Centre, Nijmegen, The Netherlands; 4Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; 5Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands; 6Department of Neurology, University of Rochester Medical Center, Rochester, MN, USA *Correspondence: Professor SM van der Maarel, Department of Human Genetics, Leiden University Medical Center, Albinusdreef 2, Leiden 2333ZA, The Netherlands. Tel: +31 715269481; Fax: +31 715268285; E-mail: [email protected] Received 30 October 2014; revised 17 February 2015; accepted 20 February 2015; published online 18 March 2015 Double SMCHD1 variants in FSHD2 ML van den Boogaard et al 79 studies showed the presence of SMCHD1 on the D4Z4 array and its For D4Z4 methylation analysis genomic DNA was double digested with reduced binding to D4Z4 in SMCHD1 mutation carriers.14 SMCHD1 EcoRI (Thermo Fisher Scientific Inc., Waltham, MA, USA) and BglII variants can also modify the disease severity in FSHD1 families, (Fermentas, Thermo Fisher Scientific) overnight at 37 °C, and cleaved DNA explaining some of the clinical variability seen in FSHD.20 was purified using PCR extraction columns (NucleoSpin Gel and PCR Clean- fi As a measure of D4Z4 chromatin relaxation, often D4Z4 methylation up, Machery-Nagel/BIOKÉ, Leiden, The Netherlands). Puri ed DNA was then ≥ is used. We have established a reliable and informative measure of digested with FseI (New England Biolabs/BIOKÉ) for 4 h, separated by size on 0.8% agarose gels, transferred to a nylon membrane (Hybond XL, D4Z4 methylation by measuring the methylation of all D4Z4 arrays Amersham, GE Healthcare, Diegem, Belgium) by Southern blotting and probed simultaneously at a unique methylation-sensitive restriction site (FseI) using the p13E-11 32P-labeled probe. Probe signals were quantified using 14 fi in the D4Z4 unit. The methylation level at this site is signi cantly the Storm 820 Phosphorimager (Amersham) and ImageQuant TL software lower in FSHD2 compared with both FSHD1 and controls, and a (Amersham). The signal from the 4061-bp fragment (methylated) was divided threshold of 25% was established for FSHD2.14 Recently, we showed by the total amount of hybridizing fragments at 4061 bp and 3387 bp that D4Z4 methylation level at this site is repeat array size (unmethylated) to yield the average percentage of methylated FseI sites within dependent.21 We introduced a new methylation parameter, Delta1, the most proximal D4Z4 unit on all four D4Z4 arrays. The Delta1 and Delta2 which represents the difference between the experimentally observed scores were calculated as described in Lemmars et al.21 methylation and the predicted methylation level based on repeat size in controls. In SMCHD1 mutation carriers, the average Delta1 score is SMCHD1 variant analysis highly negative ranging between − 20 and − 45, suggesting a strong For the index cases, SMCHD1 variant analysis was performed by Sanger fi contribution of the variant to D4Z4 hypomethylation.21 Accordingly, a sequencing after PCR ampli cation of all coding exons using intronic primers second model was then fitted to predict the methylation in SMCHD1 at a position of at least 50 nucleotides from the splice donor or acceptor site. The SMCHD1 genomic sequence was obtained from Ensemble (build 37) variant carriers, which resulted in the Delta2 score. For SMCHD1 (GRCh37:18:2655286:2805615) (Genomic Refseq: NG_031972.1, Transcript variants that preserve the open-reading frame (ORF), a mean Delta2 Refseq: NM_015295.2). Exons were numbered like in NG_031972.1 and − fi score of 1.8% was found, which is signi cantly lower than the mean primers were published previously.21 The functional consequences of variants Delta2 of ORF-disrupting variants (mean 2.7%). This suggests that were predicted using Alamut Visual version 2.4 (Interactive Biosoftware, ORF-preserving variants are more deleterious for the maintenance of a Rouen, France). Identified variants are submitted to the Leiden Open Variation repressed D4Z4 chromatin state in somatic cells.21 Database (http://databases.lovd.nl/shared/individuals/SMCHD1: submission Several studies have so far identified disease-causing variants in IDs 00028967–00028973) SMCHD1 in approximately 70 FSHD2 families. Heterozygous disease- For individual 385–203, Sanger sequencing of exons 24 and 45 in relatives causing SMCHD1 variants are distributed over the entire SMCHD1 was used to identify whether the two SMCHD1 variants were located in cis or in locus and include splice site, insertion–deletion, missense and non- trans. To identify whether the two SMCHD1 variants found in individuals – – sense variants.14,20–24 We have shown that a combination of the size of 947 201 and 1414 201 were present on different alleles, both alleles were PCR amplified and then cloned in a TOPO vector (Zero Blunt TOPO PCR Cloning the permissive D4Z4 array and the type of SMCHD1 variant together 21 Kit, Invitrogen by Life Technologies, Bleiswijk, The Netherlands). determine the epigenetic susceptibility to disease presentation. Until For individual 947–201, SMCHD1 exon 21 was PCR amplified and cloned in now, only heterozygous SMCHD1 variants, which are dominant in pCR-Blunt II-TOPO vector and transformed in DH5α heat-shock competent combination with a permissive haplotype, have been reported. Here cells (Subcloning Efficiency DH5α Competent Cells, Invitrogen, Life Technol- we describe three families in which two SMCHD1 variants that ogies). Multiple clones were analyzed, by sequencing their insert, to find clones potentially affect function were identified in each proband. For each containing either of the alleles. family, we investigated whether the variants were located in cis or For individual 1414–201, a long range PCR (Phusion High-Fidelity DNA in trans and analyzed whether both variants were contributing to Polymerase, Phusion GC Buffer Pack, New England Biolabs) from SMCHD1 D4Z4 hypomethylation independently and what the effect was on the exon 25 to exon 28 was performed (primers 25F+28R, product 8708 bp), and fi FSHD phenotype. product ends were ligated using T4 DNA Ligase (Thermo Scienti c). An additional PCR was performed using the SMCHD1 exon 28F and exon 25R primers to amplify the artificially fused exons 25 and 28; this PCR product was MATERIALS AND METHODS cloned in the pCR-Blunt II-TOPO vector and transformed in DH5α heat-shock Subjects competent cells. Multiple clones were analyzed, by sequencing their insert, to Three families were studied after informed consent and the study protocol was find clones containing either of the amplified alleles. approved by the relevant institutional review boards. Clinical assessment of disease severity was performed using the 10-point (0: unaffected; 10: wheelchair RNA analysis bound) standardized Clinical Severity Score (CSS).25 From individual 947–201, RNA was isolated from a PAXgene Blood RNA Tube The first family (Rf947) consists of a single patient living in the United States using the PAXgene Blood RNA Kit (PreAnalytix GmbH, Hombrechtikon, of America. We were not able to get additional family information from this Switzerland). cDNA was synthesized using the RevertAid First Strand cDNA individual. The second family (Rf1414) is a Dutch family, with one individual fi diagnosed with FSHD. After the identification of the two SMCHD1 variants in Synthesis Kit (Thermo Scienti c) using random hexamer primers. Reverse ′ the proband, additional clinical information and blood samples from two transcriptase PCR was performed using primers 2481F (5 -CATGGA ′ ′ ′ relatives could be collected. The third family (Rf385) is a Spanish family, with GGAAAATGGCCTTA-3 ) and 2981 R (5 -TTCAGTCGACGAGGGTGAC-3 ) one individual diagnosed with FSHD and four relatives. located in exons 18 and 23, respectively. Subsequently, PCR products were separated by size on 2% agarose gels, and PCR products were gel purified and analyzed by Sanger sequencing. D4Z4 repeat sizing, haplotype analysis and methylation analysis For genotyping high quality genomic DNA was isolated from peripheral blood RESULTS mononucleated cells. The sizing of the D4Z4 repeats on chromosomes 4 and 10 SMCHD1 in cis was done by pulsed field gel electrophoresis (PFGE) as described previously.8 Two variants in Rf947 Haplotype analysis was done by hybridization of PFGE blots with probes A and Individual 201 of Rf947 was suspected of FSHD based on physical B in combination with SSLP analysis according to previously described examination with a CSS of 7 at the age of 71 years (Figure 1).25 protocols.8 Genetic analysis showed that he carries a permissive allele of 13 D4Z4

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units and the D4Z4 methylation analysis revealed a methylation level (Figure2a).cDNAanalysisbyanRT-PCRfromSMCHD1 exon 19 to of 9% (Delta1 score − 28%), which is consistent with the diagnosis of exon 23, followed by Sanger sequencing, showed the presence of two FSHD2. SMCHD1 Sanger sequencing identified two SMCHD1 PCR products representing wild-type transcript, and the mutant variants in exon 21 in individual 947–201 (Table 1). The first variant transcript, which lacks exon 21 (Figures 2b and c). This confirms (c.2656C4T p.Arg886*) is a nonsense substitution that is predicted to that the splice donor site variant c.2700+1G4T results in a skip of result in a premature stop codon. The second variant (c.2700+1G4T) exon 21 (r.2604_2700del). Skipping of exon 21 neutralizes the is a splice donor site variant, which is predicted to result in the skip of p.Arg886* nonsense variant but will result in the disruption of the exon 21 by multiple splicing predictors (MaxEnt, NNSPLICE and ORF by a premature stop codon in exon 22. The intensity of the PCR HSF). Both variants have not been reported previously. products suggests that wild-type and mutant allele are equally To investigate whether the two variants in individual 947–201 were expressed. In contrast to previous findings where disrupting ORF located in cis or in trans, genomic DNA of SMCHD1 exon 21 was PCR variants seem to result in haploinsufficiency,21 this mutant allele might amplified and cloned. Sanger sequencing of individual exon 21 PCR not be subject to nonsense-mediated mRNA decay and possibly clones showed that both variants were located on the same allele a truncated protein will be produced.

Rf947 Rf1414 Rf385

102 101 102 62% Δ1 18% 20% Δ1 -25% 10% Δ1 -31% Δ2 7% Δ2 -1% 201 20B 22B 16B 26B 18B 27A 9% Δ1 -28% 0 78 yrs Δ2 0% 070 yrs 067 yrs c.3538G>A 13A 23B c.2941T>G c.5596C>G 7 71 yrs c.2656C>T c.2700+1G>T 201 202 202 203 206 6% Δ1 -37% 59% Δ1 8% 24% Δ1 -9% 5% Δ1 -39% 1% Δ1 -37% Δ2 -6% Δ2 -7% Δ2 -9% 22B 69A 16B 18B 13A 22B 16B 27A 26B 27A 0 53 yrs 038 yrs 7 49 yrs 842 yrs N/A 34 yrs none none c.3276_3276+4del c.2941T>G c.2941T>G c.3538G>A c.5596C>G c.5596C>G

ID % D4Z4 methylation % Delta1 (Δ1) % Delta2 (Δ2) D4Z4 units 4q allele 1 D4Z4 units 4q allele 2 Clinical severity score (CSS) Age at examination (yrs) SMCHD1 variation

Figure 1 Pedigrees of the three FSHD2 families presented in this study. Shown are families with information about D4Z4 methylation, Delta scores, sizes of 4q-linked D4Z4 repeats, clinical severity scores at the age of examination and SMCHD1 variants. Key is shown below. N/A, not available.

Table 1 SMCHD1 variants identified

Transcript position Chromosome position (NM_015295.2) Protein position Family Mutation type Position (GRCh37.p5) (NG_031972.1) (NP_056110.2) RNA analysis

Rf947 Nonsense Exon 21 g.2724949C4T c.2656C4T p.Arg886* — Rf947 5′ splice site Exon 21 g.2724994G4T c.2700+1G4T — r.2604_2700del Rf1414 5′ splice site Exon 25 g.2732490_2732494del c.3276_3276+4del —— Rf1414 Missense Exon 28 g.2740724G4A c.3538G4A p.Gly1180Arg — Rf385 Missense Exon 24 g.2729300T4G c.2941T4G p.Tyr981Asp — Rf385 Missense Exon 45 g.2784496C4G c.5596C4G p.Arg1866Gly —

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Two SMCHD1 variants in trans in Rf1414 of which one affects product was circularized and an additional PCR was performed to function amplify the fused exons 25 and 28. The PCR product was cloned in a Individual 201 of Rf1414 was suspected of FSHD based on physical TOPO vector, and individual clones were sequenced demonstrating that examination with a CSS of 7 at the age of 49 years (Figure 1). Genetic the two variants are located on different alleles (Figure 3). analysis showed that he carries a permissive allele of 13 units and the To establish whether both variants affect function, two additional D4Z4 methylation analysis revealed a methylation level of 6% (Delta1 family members were clinically evaluated and genetic analysis was − score 37%), supportive of FSHD2. performed. Sanger sequencing showed that the mother (1414–102) of fi SMCHD1 Sanger sequencing identi ed two SMCHD1 variants in individual 1414–201 carries the variant in exon 28. She shows no – individual 1414 201, one located in exon 25 (c.3276_3276+4del) and FSHD phenotype (CSS of 0 at age of 78 years) and has no FSHD 4 the other in exon 28 (c.3538G A p.Gly1180Arg) (Table 1). Deletions permissive chromosome. D4Z4 methylation analysis indicated that her in the 5′ splice site of exon 25 were previously reported in eight other D4Z4 methylation level (62%, Delta1 score 18%) is within the normal FSHD2 families suggesting it to be a mutation hotspot.21,24 Previous range (Figure 1). The sister of the index case (1414–202) does not RNA analysis of an independent FSHD2 family with a c.3276_3276 carry either of the SMCHD1 variants, no D4Z4 hypomethylation +1del variant showed that this variant results in both cryptic splicing (59%, Delta1 score 8%) and had no clinical signs of FSHD (CSS of 0 and in complete skipping of exon 25, both with retention of the – ORF.21 The same splice effect is expected for the c.3276_3276+4del at age of 53 years). Information about the father (1414 101) of – variant in individual 1414–201. The variant in exon 28 has not been individual 1414 201 was not available. This suggests that, although – reported previously. The missense predictions of SIFT and Mutation- individual 1414 201 carries two SMCHD1 variants, only the variant in Taster defined the variant in exon 28 as deleterious and disease exon 25 causes D4Z4 hypomethylation and is causal to FSHD. causing, respectively, whereas Align GVGD gives a score of C15, which indicates that is not very likely that the variant interferes with protein Two SMCHD1 variants in trans in Rf385 with additive effect function (Table 2). Individual 203 of Rf385 was suspected of FSHD based on physical To investigate whether the two variants were located on the same allele, examination with a CSS of 8 at age 42 years (Figure 1). Genetic analysis a long-range PCR of SMCHD1 exons 25–28 was performed. This PCR showed that he carries a permissive allele of 27 D4Z4 units and the

c.2656C>T p.Arg886* Exon 21 c.2700+1G>T

Allele 1 TTTGGGA TTTG AC A G CC AA GGG CCC TT G AAA C TTTT C G C AA GG C AA G TT AA

Allele 2 TTC GGGA TG TT ACCC A G AA GGG CCC T G T AAA CC T TT G T C AA GG C AA GG T AA Ref. TTC GGGA TG TT ACCC A G AA GGG CCC T G T AAA CC T TT G T C AA GG C AA GG T AA Marker Control 947-201 neg. PCR 500 bp wildtype allele 400 bp mutant allele

Wildtype TCCAAGGC C GTT TTG AAAAGC G T GGTTT ATTTTTT CC T AAAAA exon 20 exon 21

Mutant TTTCCGAAGCC G TT GG AAAAC AATTAAA T TC TGGG AA TT AC TC exon 20 exon 22

Figure 2 Two SMCHD1 variants in cis in Rf947. (a) Sanger sequence tracks from two clones showing the different SMCHD1 alleles of individual 947–201. Allele 1 contains the two variants in exon 21 at positions c.2656C4T and c.2700+1G4T, indicated with the arrows. Allele 2 contains the reference nucleotides at these positions. (b)RT-PCRofSMCHD1 with primers in exon 18 and exon 23 for a control and individual 947–201. The expected PCR product size for wild-type transcript is 491 bp; additionally, there is a smaller PCR product for 947–201, indicating an alternatively spliced mutant transcript. (c) Sanger sequence tracks of the wild-type and mutant RT-PCR products, showing skipping of exon 21 in the mutant transcript.

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Table 2 Variant predictions for identified SMCHD1 missense variants

c.3538G4A, p.Gly1180Arg c.2941T4G, p.Tyr981Asp c.5596C4G, p.Arg1866Gly

Conservation (PhyloP) Moderate (4.16) Moderate (2.38) Weak (1,09) Grantham distance Moderate (125) Large (160) Moderate (125) Align GVGD C15 (GV: 206.04–GD: 124.98) C65 (GV: 0.00–GD: 159.94) C65 (GV: 0.00–GD: 125.13) SIFT Deleterious (Score: 0) Deleterious (Score: 0) Deleterious (Score: 0) Mutation taster Disease causing (P-value: 0.999) Disease causing (P-value: 0.689) Disease causing (P-value: 0.882)

Exon 28 Exon 25 c.3538G>A p.Gly1180Arg c.3276_3276+4del

Allele 1 AAATTCCGGG AAAA T C GAATTAAG TTTA CCC T AA

Allele 2 AAATTCCGGA AAAA T C GAATTAAAGTAAGTAT CT Ref. AAATTCCGGG AAAA T C GAATTAAAGTAAGTAT CT

Figure 3 Two SMCHD1 variants in trans in Rf1414. Sanger sequence tracks from two clones showing the different SMCHD1 alleles of individual 1414–201. Allele 1 contains the variant in exon 25 (c.3276_3276+4del) and allele 2 contains the variant in exon 28 (c.3538G4A), both indicated with an arrow.

D4Z4 methylation analysis revealed a methylation level of 5% (Delta1 24, and the mother (385–102) carries the SMCHD1 variant in exon 45 score − 39%), which is consistent with the diagnosis of FSHD2. (Figure 4). This shows that both variants are located on different One of the sisters (385–206) of the index case suffered from alleles. The other sister (385–202) does not carry either of the a meningitis in childhood and she is mentally disabled; furthermore, SMCHD1 variants. she broke both her arms and a leg in the past and the neurologist was The missense predictions of SIFT and MutationTaster predict both unable to conclusively establish the clinical diagnosis of FSHD. variants as deleterious and disease causing, respectively. The Align However, she does carry one permissive allele and D4Z4 methylation GVGD scores of C65 also indicate that the variants are both likely to analysis revealed a methylation level of 1% (Delta1 score − 37%), interfere with protein function (Table 2). Additionally, both variants which is suggestive of FSHD2. are individually associated with D4Z4 methylation levels and Delta1 The other sister (385–202) does not show symptoms of FSHD with a scores in the FSHD2 range. However, D4Z4 methylation levels and CSS of 0 at age 38 years. She does carry two non-permissive alleles, and Delta1 scores are more strongly reduced in the two family members D4Z4 methylation analysis revealed a methylation level of 24% (Delta1 with both variants than in the single-variant carriers. This indicates that score − 9%), which is higher than the Delta1 range between − 20 and the effect of the variants is additive and that the combination of both − 45% observed in carriers of an SMCHD1 variantthataffects variants is more deleterious than the corresponding single variants. function.21 The father (385–101) and mother (385–102) of 385–203 do not show symptoms of FSHD, having a CSS of 0 at age 70 years and DISCUSSION CSS of 0 at age 67 years, respectively. The mother (385–102) carries one In this study, we analyzed three FSHD2 families in which two permissive allele, and D4Z4 methylation analysis revealed a methylation SMCHD1 variants that potentially affect function were found in each level of 10% (Delta1 score − 31%), suggestive of FSHD2. The father proband. Of the six SMCHD1 variants described here, one was (385–101) carries two non-permissive alleles, and D4Z4 methylation reported previously, one we demonstrated to be neutral based on analysis revealed a methylation level of 20% (Delta1 score − 25%). segregation analysis and the other four variants are novel SMCHD1 SMCHD1 Sanger sequencing identified two SMCHD1 variants in variants that affect function. 385–203 and his sister (385–206) (Figure 4). The first variant is a In individual 947–201, two variants in exon 21, separated by only 45 missense variant in exon 24 (c.2941T4G, p.Tyr981Asp), and the nucleotides, were identified on the same allele. Multiple variants in second variant is a missense variant in exon 45 (c.5596C4Gp. close proximity are seen more often and might result from chrono- Arg1866Gly) (Table 1). Both variants have not been reported coordinate events due to transient error-prone conditions.26–29 In previously. The father (385–101) carries the SMCHD1 variant in exon this case, both variants could individually be expected to affect

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Exon 24 Exon 45 c.2941T>G, p.Tyr981Asp c.5596C>G, p.Arg1866Gly

385-101 TTT CCC AAGAAAAAAT TGGA TTTTTTGGG CC GT G G

385-102 TTT CCC AAG T T A GGTT T AAAAAATTTGGG C GT C G G

385-202 TTT CCC AAG T TGGA TTTAAAAAATTTGGG CC GT G

385-203 TTT CCC AAG T TGGA TTTAAAAAATTTGGG CC GT G G G

385-206 TTT CCC AAG T TGGA TTTAAAAAATTTGGG CC GT G G G Ref. TTT CCC AAG T T A GGTT T AAAAAATTTGGG CC GT G

Figure 4 Two SMCHD1 variants in trans in Rf385. Sanger sequence tracks from Rf385 family members showing that the two variants are located in trans. Individuals 385–203 and 385–206 carry both SMCHD1 variants (c.2941T4G and c.5596C4G), both indicated with an arrow. Individual 385–101 carries only the SMCHD1 variant in exon 24. Individual 385–102 carries only the SMCHD1 variant in exon 45. Individual 385–202 carries the reference nucleotide for both variants.

function. The first variant (c.2656C4T) is a nonsense substitution, would be present independently, it would also disrupt the ORF, which will cause reading frame interruption by a premature stop probably resulting in hypomethylation and an FSHD phenotype. codon. The second variant (c.2700+1G4T) is located in the splice In individual 1414–201, two variants on different alleles were donor site of exon 21, and this variant is predicted to cause skipping of identified. Although the missense substitution in exon 28 was exon 21. mRNA analysis revealed that the splice donor site variant predicted to affect function by SIFT and MutationTaster (but not by c.2700+1G4T indeed results in a skip of exon 21. The skipping of Align GVGD), segregation analysis showed that this variant does not exon 21 will disturb the ORF, resulting in a premature stop codon in independently cause D4Z4 hypomethylation, and is therefore likely a exon 22. The intensity of the mutant transcript on gel indicates that neutral variant, even on a FSHD-permissive background. The second this may not lead to nonsense-mediated mRNA decay, but we were variant found in individual 1414–201 is a splice donor site variant in unable to study the variant at the protein level. The mean Delta2 score exon 25. Splice donor site variants in exon 25 have already been for ORF-disrupting variants is 2.7%, while it is − 1.8% for ORF- reported in eight other FSHD2 families to segregate with D4Z4 preserving variants.21 The Delta2 score of 947–201 is 0%, which in this hypomethylation and disease presentation, confirming the functional case is inconclusive. consequences of this variant.21,24 With the skipping of exon 21, the nonsense substitution In individual 385–203, two variants on different alleles were c.2656C4T has no effect on the transcript. However, if this variant detected. Both variants are predicted to affect function by SIFT,

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MutationTaster and Align GVGD. In accordance, both variants cause FSHD2. In one of the three families, both variants contribute D4Z4 hypomethylation independently, as observed in the parents of separately to the disease, as is reflected by the Delta1 and Delta2 385–203. In this family, the combination of both variants further scores in this family and the penetrance of the disease. Importantly, decreases the Delta1 score at D4Z4 from − 25% in the father this family shows that a combination of two ORF-preserving variants (385–101) and − 31% in the mother (385–102), who both carry one in SMCHD1 is compatible with life. SMCHD1 variant, to − 39% in the patient (385–203) and − 37% in his sister (385–206), both double SMCHD1 variant carriers (Figure 1). CONFLICT OF INTEREST This suggests a synergistic effect of both variants. The other sister The authors declare no conflict of interest. (202) has a Delta1 score of − 9%, indicating that she has moderate D4Z4 hypomethylation, independent of an SMCHD1 variant. There- ACKNOWLEDGEMENTS fore, the data are consistent with an additive effect of having one or We thank the families for participating in our studies. This study was supported two SMCHD1 missense variants on D4Z4 methylation. This additive with grants from the US National Institutes of Health (NIH) (National Institute effect is also shown by the Delta2 scores, which are reduced in 385– of Neurological Disorders and Stroke (NINDS) P01NS069539, and National 203 (−7%) and 385–206 (−9%), who carry both missense variants, Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) compared with the father (7%) and mother (−1%). R01AR045203), the Muscular Dystrophy Association (MDA; 217596), the The synergistic effect of both variants also explains the very low Fields Center for FSHD Research, the Geraldi Norton and Eklund family – D4Z4 methylation levels and severe FSHD phenotype of individual foundation, the FSH Society, The Prinses Beatrix Spierfonds (W.OR12 20), Spieren voor Spieren, The Friends of FSH Research and European Union 385–203. It is unfortunate that it is not possible to establish whether Framework Programme 7 agreement 2012–305121 (NEUROMICS). the sister 385–206 is affected with FSHD. The father (385–101) carries two non-permissive D4Z4 alleles, which explains why he is not affected with FSHD. With regards to the mother (385–102), the size of her permissive allele (27 units) might explain why she is not affected 1 Deenen JC, Arnts H, van der Maarel SM et al: Population-based incidence with FSHD, despite her SMCHD1 variant and D4Z4 hypomethylation. and prevalence of facioscapulohumeral dystrophy. Neurology 2014; 83: In 2010, de Greef et al4 showed that the average size of the shortest 1056–1059. 2 Padberg GW, Lunt PW, Koch M, Fardeau M: Diagnostic criteria for facioscapulohumeral permissive allele in FSHD2 is 16 units, which is much shorter than the muscular dystrophy. Neuromuscul Disord 1991; 1:231–234. average of 28 units found in control individuals, which was confirmed 3 Statland JM, Tawil R: Facioscapulohumeral muscular dystrophy: molecular pathological in a later study.4,21 The permissive allele in the proband (385–203) and advances and future directions. Curr Opin Neurol 2011; 24:423–428. – 4 de Greef JC, Lemmers RJ, Camano P et al: Clinical features of facioscapulohumeral his mother (385 102) is 27 units, relatively long for FSHD2. This muscular dystrophy 2. Neurology 2010; 75: 1548–1554. might partially explain why the mother does not show an FSHD 5 van Overveld PG, Lemmers RJ, Sandkuijl LA et al: Hypomethylation of D4Z4 in phenotype and why two SMCHD1 variants are necessary in this family 4q-linked and non-4q-linked facioscapulohumeral muscular dystrophy. Nat Genet 2003; 35:315–317. to present FSHD symptoms. However, it is also known that there are 6 ZengW,deGreefJC,ChenYYet al: Specific loss of histone H3 lysine 9 trimethylation FSHD2 patients carrying a single SMCHD1 variant and a single and HP1gamma/cohesin binding at D4Z4 repeats is associated with facioscapulohum- 21 eral dystrophy (FSHD). PLoS Genet 2009; 5: e1000559. permissive allele of as much as 40 units. 7 Snider L, Geng LN, Lemmers RJ et al: Facioscapulohumeral dystrophy: incomplete Furthermore, variability in clinical representation is a hallmark of suppression of a retrotransposed gene. PLoS Genet 2010; 6: e1001181. FSHD, which is seen both within and between families, and more often 8 Lemmers RJ, van der Vliet PJ, Klooster R et al: A unifying genetic model for – 30–32 facioscapulohumeral muscular dystrophy. Science 2010; 329:16501653. in females than in males. Some, but not all, of this variability can 9 Balog J, Thijssen PE, de Greef JC et al: Correlation analysis of clinical parameters be explained by the size of the permissive D4Z4 repeat and presence with epigenetic modifications in the DUX4 promoter in FSHD. Epigenetics 2012; 7: and type of an SMCHD1 variant.21 Both variants in this family are 579–584. 10 Bosnakovski D, Xu Z, Gang EJ et al: An isogenetic myoblast expression screen identifies ORF-preserving variants, which have in general a more profound effect DUX4-mediated FSHD-associated molecular pathologies. EMBO J 2008; 27:2766–2779. on D4Z4 methylation level than ORF-disrupting variants.21 11 Kowaljow V, Marcowycz A, Ansseau E et al: The DUX4 gene at the FSHD1A locus encodes a pro-apoptotic protein. Neuromuscul Disord 2007; 17:611–623. In the mouse, Smchd1 has a role in the establishment and 12 Geng LN, Yao Z, Snider L et al: DUX4 activates germline genes, retroelements, and maintenance of CpG methylation of a subset of genes on the inactive immune mediators: implications for facioscapulohumeral dystrophy. 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Nat Rev Mol Cell Biol 2006; 7:311–322. function and a neutral variant, in addition to the approximately 70 19 Ball ARJr., Chen YY, Yokomori K: Mechanisms of cohesin-mediated gene regulation variants that already have been identified since the discovery of and lessons learned from cohesinopathies. Biochim Biophys Acta 2014; 1839: 191–202. SMCHD1 as the most common FSHD2 gene in 2012. This is the 20 Sacconi S, Lemmers RJ, Balog J et al: The FSHD2 gene SMCHD1 is a modifier first report of families with individuals carrying two SMCHD1 variants of disease severity in families affected by FSHD1. Am J Hum Genet 2013; 93: and this study highlights the usefulness of D4Z4 methylation analysis 744–751. 21 Lemmers RJ, Goeman JJ, van der Vliet PJ et al: Inter-individual differences in CpG to determine the functional consequences of SMCHD1 variants. These methylation at D4Z4 correlate with clinical variability in FSHD1 and FSHD2. 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European Journal of Human Genetics Double SMCHD1 variants in FSHD2 ML van den Boogaard et al 85

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